The bone marrow provides a unique environment for multipotential and committed cells. It contains both structural and humoral components that have yet to be successfully duplicated in culture. The marrow cavity itself is a network of thin-walled sinusoids lined with endothelial cells. Between the walls of bone are clusters of hematopoietic cells and fat cells constantly fed by mature blood cells entering through the endothelium. Differentiated cells ready to function within the circulatory system depart the cavity in a similar fashion.
Hematopoietic stem cells (HSC) are the most primitive cells of the hematopoietic lineage, and have the ability to give rise to all cells of the hematopoietic lineage (including HSC). HSC are known to reside in the bone marrow, but their specific niche within the bone marrow microenvironment is not currently defined. Previous studies have established that certain HSC progeny, the lineage-restricted clonogenic hematopoietic progenitor cells (HPC), conform to a well-defined spatial distribution across the axis of the femur with greatest numbers near the central longitudinal vein. Such observations foster the widely held belief that the distinct spatial organization exhibited by these various cell populations within the bone marrow is a manifestation of specific adhesive interactions occurring with the underlying stromal tissue. However, due to the rarity of HSC and the lack of a single, unique antigenic marker allowing their unambiguous identification in situ, it has not been possible to define the spatial distribution of HSC within the bone marrow.
Evidence now exists to suggest that hematopoiesis is localized to the bone marrow by developmentally regulated adhesive interactions between primitive HSC and the stromal cell mediated microenvironment. It is likely that the adhesive interactions in this microenvironment serve multiple functions, including homing and lodgement of HSC to the bone marrow during ontogeny or following transplantation, and participation in the direct regulation of their proliferation and differentiation.
The extracellular matrix (ECM) is the major component of connective tissue which provides structural integrity, and promotes cell migration and differentiation. As part of these functions, extracellular matrix molecules such as fibroncetin, collagen, laminin, fibrinogen, and tenascin have been shown to support adhesion of cells in vitro. This adhesive interaction is critical for a number of biological processes including hemostasis, thrombosis, wound healing, tumor metastasis, immunity and inflammation.
A class of receptors involved with mediation of adhesive interaction with extracellular matrix molecules are the integrins, which consist of heterodimeric complexes of non-covalently associated alpha and beta subunits. A common β subunit combines with unique α subunits to form an adhesion receptor of defined specificity. The β1 subfamily, also known as the VLA family (Very Late Activation Antigens), binds to ECM molecules such as FN, collagen and laminin. For reviews, see, Hynes, Cell 48:549 (1987); Hemler, Annu. Rev. Immunol. 8:365 (1990).
Bone marrow transplantation is a useful treatment for a variety of hematological, autoimmune and malignant diseases, where there is a need to replenish hematopoietic cells of the bone marrow (via hematopoiesis) that have been depleted by treatments such as chemotherapy and radiotherapy. Current bone marrow transplantation therapies include the use of hematopoietic cells obtained from umbilical cord blood or from peripheral blood (either unmobilized or mobilized with agents such as G-CSF), as well as directly from the bone marrow.
A limitation in bone marrow transplantation is obtaining enough stem cells to restore hematopoiesis. Current therapies may include the ex vivo manipulation of hematopoietic cells to expand primitive stem cells to a population suitable for transplantation. Moreover, whilst there is rapid regeneration to normal pre-transplantation levels in the number of hematopoietic progenitors and mature end cells following bone marrow transplantation, HSC numbers recover to only 5-10% of normal levels. The available methodologies do not adequately address ex vivo HSC manipulation, and thus the cell populations used in clinical applications are limited by the number of cells that are able to be isolated from the donor. For example, due to the limited number of multipotential HSC in umbilical cord blood, cells from this source can only be used for transplantation in younger patients, and excludes the adult population in need of HSC transplantation therapies.
In addition to issues impacting upon therapeutic uses, there exists the problem of obtaining sufficient numbers of HSC for clinical studies, drug development, or research purposes. An understanding of HSC activity and behaviour is tremendously important in improving the efficacy of therapies, and in determining the toxicity of various therapeutics. Isolation of normally occurring populations of stem or progenitor cells in adult tissues has been technically difficult and costly, due, in part, to the limited quantity of stem or progenitor cells found in blood or tissue, and the significant discomfort involved in obtaining bone marrow aspirates. In general, harvesting of stem or progenitor cells from alternative sources in adequate amounts for therapeutic and research purposes is generally laborious, the sources are limited due to the nature of the harvesting procedures, and the yield is low.
There is therefore a need to provide a method for isolating a cell population enriched in HSCs.